The infectious yeast Candida albicans remains an important concern in public health, particularly with immune-compromised individuals. One set of virulence factors for C. albicans includes a family of copper containing superoxide dismutase (SOD) enzymes that scavenge toxic superoxide free radicals. In spite of their established importance in virulence, virtually nothing is known about the biochemistry of C. albicans SODs or how these enzymes are activated in cells through insertion of the copper co-factor. In this research application, we plan to develop new systems for monitoring the effects of the host on intracellular copper and copper containing SODs of C. albicans . Two types of Cu-containing SODs in C. albicans will be examined: a Cu/Zn SOD in the cytosol (SOD1), and an extracellular member of the Cu/Zn SOD family attached to the cell wall through GPI anchors (SOD5). Both are important for virulence and in our preliminary analyses, both exhibit some distinctive features not seen in other Cu/Zn SODs. For example, under normal (non-infectious) laboratory growth conditions, the intracellular SOD1 protein is inactive and the yeast instead uses an unusual cytosolic manganese-SOD3 to remove superoxide. The extracellular SOD5 is also unique in that it appears to bind only copper, and is lacking the zinc site that is invariant among other eukaryotic Cu/Zn SODs. We propose that these unusual Cu-SODs have evolved to exploit the host effects on metals. During infection, the macrophage phagolysosome starves pathogens of manganese, zinc and iron, while attempting to kill pathogens with high levels of copper and reactive oxygen. This particular environment of the host seems ideal for activating copper requiring SODs. We hypothesize that during infection, both the intracellular SOD1 and extracellular SOD5 are immediately charged with copper, representing the first line of defense against the oxidative burst of the host. We will begin to test this hypothesis by developing systems in which we can monitor changes in intracellular copper and Cu/Zn SOD activity that occur in C. albicans during infection of macrophages. We will also develop a system for recovering enzymatically active SOD5 from the cell wall or secreted from yeast, a tool that will prove invaluable for monitoring extracellular SOD activation during macrophage infection. Through metal analysis of recombinant SOD5, we will address whether this enzyme is truly a copper-only SOD. These studies are in the exploratory, assay-development phase. The findings and new systems that stem from this 2 year program will foster our more long-term mechanistic studies geared towards understanding host effects on pathogen metals and SOD enzymes. These basic research studies have the potential to seed development of new therapies that target copper and prevent activation of the C. albicans SODs essential for virulence.